*Target (protein/gene name):enoyl-acyl carrier reductase *NCBI Gene # or RefSeq#: 7901395 *Protein ID (NP or XP #) or Wolbachia#: AAQ74987.1 *Organism (including strain): Toxoplasma gondii Etiologic Risk Group: Risk Group 2 Parasitic Agent *Background/Disease Information:
Toxoplasma gondii is caused by the protozoan parasite. It is the most widespread parasitic infections and leads to congenital neurological birth dects. It is the third most common cause of food-borne deaths in the US. It infects 25% of the world population, usually through consumption of undercooked contaminate meat. No drugs to date have been made available to provide a fully effective treatment. Efforts have been made to interact with the enoyl reductase, which leadsto the inhibition of the fatty-acid biosynthesis pathway in the protozoan.
Essentiality of this protein: The protein is essential in fatty-acid biosynthesis in the T. gondii. Without the enzyme, the protozoan lacks a crucial biomolecule. Complex of proteins: In complex with NAD and triclosan Druggable Target: 0.8; TCL ligand [triclosan]
Structure Available (PDB or Homology model) -- PDB # or closest PDB entry if using homology model: 2O2S -- For Homology Model option: ---- Show pairwise alignment of your BLASTP search in NCBI against the PDB
---- Query Coverage: 75% ---- Max % :100% ---- % Positives: 100% ---- Chain used for homology: 2O2S from PDB and NCBI protein AAQ74987.1
Current Inhibitors: antibacterial agent triclosan Expression Information (has it been expressed in bacterial cells): Yes, it is expressed in BL21 E. Col, combined with the pMALc2x vector. Cells were grown in LB medium at 310 K to an optimal density. Purification Method:
Cells were resuspended in a lysis buffer. The buffer contains 20mM sodium/potassium phosphate, 20mM NaCl, and 2.5 micrograms of DNAse I.The lysate is
centrifuged and applied onto a HiTrap Chelating HP column. TgENR (the protein) was eluted in a gradient and then desalted. The TgENR was then pooled and
passed through a substractive amylose column to remove any residual fusion proteins, leaving us with pure TgENR fractions.
Image of protein (PyMol with features delineated and shown separately):
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only): *GC% Content for gene (codon optimized):
Don't need- Primer design results for pNIC-Bsa4 cloning (list sequences of all of your ~40 nt long primers): (link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol) -- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences): Resources:
*NCBI Gene # or RefSeq#: 7901395
*Protein ID (NP or XP #) or Wolbachia#: AAQ74987.1
*Organism (including strain): Toxoplasma gondii
Etiologic Risk Group: Risk Group 2 Parasitic Agent
*Background/Disease Information:
Toxoplasma gondii is caused by the protozoan parasite. It is the most widespread parasitic infections and leads to congenital neurological birth dects. It is the third most common cause of food-borne deaths in the US. It infects 25% of the world population, usually through consumption of undercooked contaminate meat. No drugs to date have been made available to provide a fully effective treatment. Efforts have been made to interact with the enoyl reductase, which leadsto the inhibition of the fatty-acid biosynthesis pathway in the protozoan.
Essentiality of this protein: The protein is essential in fatty-acid biosynthesis in the T. gondii. Without the enzyme, the protozoan lacks a crucial biomolecule.
Complex of proteins: In complex with NAD and triclosan
Druggable Target: 0.8; TCL ligand [triclosan]
*EC#: 1.3.1.9
Link to BRENDA EC# page:
http://www.brenda-enzymes.org/php/result_flat.php4?ecno=1.3.1.9&Suchword=&organism%5B%5D=&show_tm=0
-- Show screenshot of BRENDA enzyme mechanism
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents):
-- link to Sigma (or other company) page for assay or assay reagents (substrates)
http://www.sigmaaldrich.com/catalog/search?interface=All&term=toxoplasma%20gondii%20reductase&lang=en®ion=US&focus=product&N=0+220003048+219853269+219853286
)
-- link (or citation) to paper that contains assay information
http://www.sigmaaldrich.com/catalog/papers/10779791
-- List cost and quantity of substrate reagents and supplier
Substrate reagent: sulfadoxin [S7821]
10G = $64.50
25G = $158.50
Supplier: Sigma-Aldrich
http://www.sigmaaldrich.com/catalog/product/sigma/s7821?lang=en®ion=US&cm_sp=abstract-_-10779791-_-S7821
Structure Available (PDB or Homology model)
-- PDB # or closest PDB entry if using homology model: 2O2S
-- For Homology Model option:
---- Show pairwise alignment of your BLASTP search in NCBI against the PDB
Query 103 SAFPIDLRGQTAFVAGVADSHGYGWAIAKHLASAGARVALGTWPPVLGLFQKSLQSGRLD 162 SAFPIDLRGQTAFVAGVADSHGYGWAIAKHLASAGARVALGTWPPVLGLFQKSLQSGRLD Sbjct 1 SAFPIDLRGQTAFVAGVADSHGYGWAIAKHLASAGARVALGTWPPVLGLFQKSLQSGRLD 60 Query 163 EDRKLPDGSLIEFAGVYPLDAAFDKPEDVPQDIKDNKRYAGVDGYTIKEVAVKVKQDLGN 222 EDRKLPDGSLIEFAGVYPLDAAFDKPEDVPQDIKDNKRYAGVDGYTIKEVAVKVKQDLGN Sbjct 61 EDRKLPDGSLIEFAGVYPLDAAFDKPEDVPQDIKDNKRYAGVDGYTIKEVAVKVKQDLGN 120 Query 223 IDILVHSLANGPEVTKPLLETSRKGYLAASSNSAYSFVSLLQHFGPIMNEGGSAVTLSYL 282 IDILVHSLANGPEVTKPLLETSRKGYLAASSNSAYSFVSLLQHFGPIMNEGGSAVTLSYL Sbjct 121 IDILVHSLANGPEVTKPLLETSRKGYLAASSNSAYSFVSLLQHFGPIMNEGGSAVTLSYL 180 Query 283 AAERVVPGYGGGMSSAKAALESDTRTLAWEAGQKYGVRVNAISAGPLKSRAASAIGKSGE 342 AAERVVPGYGGGMSSAKAALESDTRTLAWEAGQKYGVRVNAISAGPLKSRAASAIGKSGE Sbjct 181 AAERVVPGYGGGMSSAKAALESDTRTLAWEAGQKYGVRVNAISAGPLKSRAASAIGKSGE 240 Query 343 KSFIDYAIDYSYNNAPLRRDLHSDDVGGAALFLLSPLARAVSGVTLYVDNGLHAMGQAVD 402 KSFIDYAIDYSYNNAPLRRDLHSDDVGGAALFLLSPLARAVSGVTLYVDNGLHAMGQAVD Sbjct 241 KSFIDYAIDYSYNNAPLRRDLHSDDVGGAALFLLSPLARAVSGVTLYVDNGLHAMGQAVD 300 Query 403 SRSMPPLQRATQEIN 417 SRSMPPLQRATQEIN Sbjct 301 SRSMPPLQRATQEIN 315---- Query Coverage: 75%
---- Max % :100%
---- % Positives: 100%
---- Chain used for homology: 2O2S from PDB and NCBI protein AAQ74987.1
Current Inhibitors: antibacterial agent triclosan
Expression Information (has it been expressed in bacterial cells): Yes, it is expressed in BL21 E. Col, combined with the pMALc2x vector. Cells were grown in LB medium at 310 K to an optimal density.
Purification Method:
Cells were resuspended in a lysis buffer. The buffer contains 20mM sodium/potassium phosphate, 20mM NaCl, and 2.5 micrograms of DNAse I.The lysate is
centrifuged and applied onto a HiTrap Chelating HP column. TgENR (the protein) was eluted in a gradient and then desalted. The TgENR was then pooled and
passed through a substractive amylose column to remove any residual fusion proteins, leaving us with pure TgENR fractions.
Image of protein (PyMol with features delineated and shown separately):
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
*length of your protein in Amino Acids: 315
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website: 2.008635797e+25 kDa
Molar Extinction coefficient of your protein at 280 nm wavelength: 35870 M−1cm−1
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
*CDS Gene Sequence (paste as text only):
forward 5′-GGTGGTGAATTCTCAAACATAAACAAAATTAAAGAAG-3′
reverse 5′-GGTGGTGTCGACTTATTCATTTTCATTGCGATATATATC-3′
Primers amplified nucleotides encoding amino-acid residues 103–417 of TgENR
http://www.brenda-enzymes.org/literature/lit.php4?e=1.3.1.9&r=671150
*GC% Content for gene: 50.3%
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
*GC% Content for gene (codon optimized):
Don't need-
Primer design results for pNIC-Bsa4 cloning (list sequences of all of your ~40 nt long primers):
(link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
Resources:
See ProtocolTargetDiscoveryVDS.docx for more
Etiologic Risk Group Categories (for pathogens): http://www.utexas.edu/research/rsc/ibc/agent_class.html#_Toc7238334
Databases of genes/organisms:
http://tdrtargets.org/
Scientific Nomenclature page from Center for Disease Control (gene, protein names and abbreviations)
http://wwwnc.cdc.gov/eid/pages/scientific-nomenclature.htm
Gene Information:
NCBI GENE Page: http://www.ncbi.nlm.nih.gov/gene
BLAST Page: http://blast.ncbi.nlm.nih.gov/
Protein Information:
NCBI Protein Page: http://www.ncbi.nlm.nih.gov/protein
Protein Expression Website
Protein Expression Paper: SGC_ProteinProductionPurificationNatMethods2008.pdf
Primer Overlap PCR Articles
HooverLubkowski_PCRoverlapcloninggnf042.pdf
StemmerPCRoverlapGene1995.pdf
http://www.sciencedirect.com/science/article/pii/S0014579300021281
http://www.brenda-enzymes.org/literature/lit.php4?e=1.3.1.9&r=671150
http://www.brenda-enzymes.org/literature/lit.php4?e=1.3.1.9&r=712708